We describe here a plan to implement a high throughput screen (HTS) for inhibitors of the Nox1 NADPH oxidase. Nox1 is a member of a recently identified 7 member family of NADPH oxidases that plays important roles in cell signaling, growth, motility, and host defense. Currently, no specific inhibitors of NADPH oxidases in general, nor of Nox1 specifically, have been identified. The identification of a small molecule inhibitor of Nox1 would be extremely useful for studies into the biology of the Nox enzymes. In addition, such an inhibitor could represent a novel therapeutic approach to colon cancer, hypertension, and inflammatory diseases. We have developed a cell- based assay of Nox1 function that is now ready for a large scale HTS in conjunction with the MLSCN. The Specific Aims for this proposal are: 1. Implement the Nox1 screen at the nearby MSLCN center. We will participate in transferring the assay to a screening center such that ~105 to 106 compounds in the MLSCN libraries can be screened. The cell-based assay, which uses luminol to detect Nox1- dependent superoxide formation, has been fully validated and adapted to use in 384-well plates. Statistical analysis of the Nox1 assay confirms that it has a useful signal window that exceeds requirements for a validated screen (Z factor of 0.68). 2. Validate the primary hits from the MSLCN screen with followup assays. A series of followup assays are in place with increasing levels of rigor for the primary hits that emerge from the screening. First, the hits will be verified with full scale cellular assay using transfected Nox1 in which ROS formation is determined using a homovannilic acid-based peroxide assay.. Dose-response curves will be obtained to insure specific saturable inhibitor activity. Third, inactivity against other oxidative enzymes will be verified. Fourth, hits will be tested against a series of cells expressing other Nox family members to evaluate the Nox family selectivity of the inhibitors. 3. Use the validated inhibitors to probe the biological function and regulation of Nox1 using biochemical and genetic tools in biological assays. The compounds that emerge as validated hits will be powerful investigative tools into the biology and regulation of Nox1 when used in combination with ongoing biochemical and functional analyses. The effect of inhibitors on the interactions of Nox1 with known cytosolic regulators will be studied. The action of Nox1 in inflammatory responses of the colon and in colon cancer will be studied. PUBLIC HEALTH RELEVANCE: It is known that NADPH oxidases (Nox) play important roles in health and disease, yet much more remains to be learned about Nox1 function in biology. We will use a cell-based screen to identify Nox1 inhibitors, followed by secondary screens to verify selectivity, efficacy and mechanism. Such inhibitors will be useful to investigate Nox1 biology, and as potential therapeutic agents in inflammatory diseases and cancer. [unreadable] [unreadable] [unreadable]